Multiplex assays with multiple luciferases reporters and uses thereof

ABSTRACT

The present invention encompasses modified luciferases, methods for making modified luciferases, and assays utilizing modified luciferases. Modified luciferases of the invention show increased activity over wildtype luciferases and also show increased stability of signal. The present invention also encompasses multiplex assays utilizing multiple luciferases reporters with different emission spectra and different substrates for simultaneous luciferase measurements.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 61/238,146, filed on Aug. 29, 2009, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention concerns the field of luciferase reporters useful in biological and biochemical assays.

BACKGROUND OF THE INVENTION

Luciferases are enzymes that catalyze reactions that emit light. Luciferases are named according to their source organisms such as beetles (firefly) or marine organisms. Examples of bioluminescent marine animals include: Renilla, also known as sea pansies, which belong to a class of coelenterates known as the anthozoans. In addition to Renilla, other representative bioluminescent genera of the class Anthozoa include Cavarnularia, Ptilosarcus, Stylatula, Acanthoptilum, and Parazoanthus. All of these organisms are bioluminescent and emit light as a result of the action of an enzyme (luciferase) on a substrate (luciferin) under appropriate biological conditions.

Different luciferases have different properties with regard to substrate specificity and intensity of light emission and stability of the bioluminescent signal, which is commonly measured by a luminometer. Luciferases are useful as transcriptional reporter genes and in imaging reporter gene expression in living subjects and many other applications in molecular biology.

Certain luciferases, such as those that utilize cypridina luciferin (vargulin) as a substrate, can be useful reporters because of their strong luminescent signal and the fact that they are secreted in the native form. However cypridina luciferin (vargulin) is very difficult to synthesize (usually involving an 18-step chemical synthesis). The limiting supply and the cost of the material have made the assay difficult to commercialize.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides modified luciferases, methods of making modified luciferases, and methods of using modified luciferases.

In one aspect, the present invention provides an isolated polynucleotide that encodes a modified Luciola Italica (also referred to as L. Italica) luciferase. In a further aspect, the modified L. Italica luciferase shows increased luciferase activity when expressed in mammalian cells as compared to a non human codon optimized mutant L. Italica luciferase.

In an embodiment and in accordance with any of the above, the present invention provides a modified L. Italica luciferase that shows an approximately 1000-fold increased luciferase activity when expressed in mammalian cells as compared to a non human codon optimized mutant L. Italica luciferase.

In a further embodiment and in accordance with any of the above, the present invention provides a modified L. Italica luciferase that is a red-emitting luciferase with an emission maximum of approximately 617 nm.

In a further embodiment and in accordance with any of the above, the present invention provides a modified L. Italica luciferase that is human codon-optimized.

In a further embodiment and in accordance with any of the above, the present invention provides a modified L. Italica luciferase that is a green-emitting luciferase with an emission maximum of approximately 550 nm.

In a further embodiment and in accordance with any of the above, the present invention provides a modified L. Italica luciferase that includes a secretory signal at its amino terminal end. In a still further embodiment, the secretory signal is a chymotrypsinogen secretory signal.

In one aspect, the present invention provides assays utilizing any of the modified luciferases discussed herein. In a further aspect, the assays are multiplexed reporter assays.

In one aspect, the present invention provides an isolated polynucleotide that encodes a modified Renilla luciferase. In a further aspect, the modified Renilla luciferase shows increased activity and stability over a native human codon optimized Renilla luciferase.

In an exemplary embodiment the present invention provides a modified Renilla luciferase that is a green-emitting Renilla luciferase.

In a further embodiment and in accordance with any of the above, the invention provides a modified Renilla luciferase that includes a secretory signal at its amino terminal end.

In one aspect, the present invention provides multiplexed luciferase assays comprising at least two different luciferase reports, where the at least two different luciferase reporters emit at two different wavelengths and/or utilize different substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows data for relative luciferase stability for a Cypridina assay conducted using reagents without sodium chloride (VLAR-1) and with sodium chloride (VLAR-1 with sodium chloride).

FIG. 2 shows data for time course of activity in a Cypridina assay using 5 μl of sample (FIG. 2A) or 20 μl of sample (FIG. 2A).

FIG. 3A-B shows the sequence of a green Renilla luciferase plasmid (SEQ ID NO: 1).

FIG. 4 shows the sequence of a modified red firefly luciferase with a secretory signal (SEQ ID NO: 2).

FIG. 5 shows data from a Cypridina luciferase assay in varying concentrations of sodium chloride.

FIG. 6 shows data from a Renilla luciferase assay with and without stabilizer (NP40).

FIG. 7 shows data comparing luciferase activity of native human codon optimized Renilla luciferase and a mutant Renilla luciferase of the invention.

FIG. 8 shows data comparing luciferase activity of human codon optimized and non-human codon optimized red-emitting L. Italica luciferase.

FIG. 9 shows data comparing luciferase activity of human codon optimized and non-human codon optimized green-emitting L. Italica luciferase.

FIG. 10 shows data comparing luciferase activity of intracellular red-emitting L. Italica luciferase and secreted red-emitting L. Italica luciferase.

FIG. 11 shows data comparing luciferase activity of secreted red L. Italica luciferase in the lysate and the supernatant from HEK293 cells.

FIG. 12 shows kinetics of luciferase activity in (A) Red Luciola luciferase, (B) Guassia luciferase, (C) Cypridina luciferase, and (D) Green Renilla luciferase.

FIG. 13 shows emission spectra from (A) a double reporter assay with Vargula and Red Italica luciferases and (B) a triple reporter assay with Vargula, Green Renilla and Red Italica Luciferases.

FIG. 14 shows kinetics data of a Gaussia luciferase assay using a GAR-1 reagent.

FIG. 15 shows data comparing stabilities of Gaussia luciferase assays using the GAR-2 reagent are in the presence of a stabilizer (FIG. 15A) and in the absence of a stabilizer (FIG. 15B).

FIG. 16 shows data related to relative luciferase activity of a firefly luciferase assay.

FIG. 17 shows data of relative luciferase activity of a Cypridina luciferase assay.

FIG. 18 shows data comparing luciferase activity of a modified Vargula luciferase of the invention in the lysate and the supernatant from mammalian cells.

FIG. 19 shows kinetic data for luciferase activity in a Cypridina luciferase assay.

FIG. 20 shows data comparing relative luciferase activity of Green Renilla luciferase in the absence (top panel) and presence (bottom) of a stabilizer.

FIG. 21 shows data from a firefly luciferase assay in the presence (square) and absence (diamonds) of a stabilizer.

FIG. 22 shows data from a dual assay of the invention utilizing firefly and Cypridina luciferases.

FIG. 23 shows data from a dual assay of the invention utilizing Cypridina (Panel A) and Renilla (Panel B) luciferases.

FIG. 24 shows emission spectra from a dual assay of the invention utilizing Vargula and Green Renilla luciferases.

FIG. 25 shows data from a triple assay of the invention utilizing Cypridina, firefly and Gaussia luciferases.

FIG. 26 shows emission spectra from a triple assay of the invention utilizing Cypridina, Green Renilla and Red Italica luciferases.

FIG. 27 shows the sequence of a red firefly luciferase of the invention (SEQ ID NO: 5).

FIG. 28 shows emission spectra from a dual assay of the invention utilizing Vargula and Red Italica luciferases.

FIG. 29 shows emission spectra emission spectra from a dual assay of the invention utilizing (A) Gaussia and Red Italica luciferases and (B) Green Renilla and Red Italica luciferases.

FIG. 30 shows the sequence of a red emitting firefly human codon optimized luciferase of the invention (SEQ ID NO: 3).

FIG. 31 shows the sequence of a human codon optimized green firefly luciferase of the invention (SEQ ID NO: 4).

FIG. 32 shows the sequence of a human codon optimized Vargula luciferase of the invention (SEQ ID NO: 6).

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, formulations and methodologies which are described in the publication and which might be used in connection with the presently described invention.

Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polymerase” refers to one agent or mixtures of such agents, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. It will be apparent to one of skill in the art that these additional features are also encompassed by the present invention.

Overview

The present invention provides modified luciferases and/or combinations of luciferases, and methods of utilizing those luciferases in reporter gene assays. In addition, the invention provides reagents that provide increased stability and activity in assays using luciferase reporters.

The present invention provides modified (also referred to herein as “mutant” or “variant”) luciferases showing improved activity over wildtype luciferases or other modified luciferases known in the art reported to have improved properties for reporter gene assays or in vivo imaging applications. As used herein, “wildtype luciferases” refers to any luciferase that occurs in nature.

In certain aspects, the present invention provides modified luciferases that show brighter luminescence when expressed in mammalian cells as compared to the luminescence seen when wildtype luciferases are expressed in mammalian cells. The present invention also provides a method of expressing luciferase as a very bright intracellular reporter (not secreted) by sequence modification to increase its utility as an intracellular reporter in multiplexed assays and for imaging applications. The present invention also provides a composition for assays utilizing luciferases that lowers the cost and increases the efficiency and sensitivity of the assay by altering the reaction conditions such that high luminescence is produced using significantly less amount of luciferin.

The present invention further provides reagents for assays utilizing modified luciferases of the invention as well as mammalian expression vectors expressing secreted and intracellular luciferases.

In further embodiments the present invention provides sequence modifications (human codon optimization) to nucleotides encoding luciferases which result in an approximately 1000-fold increase in luciferase expression in transfected mammalian cells compared to the non-human codon optimized versions of these genes.

In further embodiments, the invention provides novel secreted reporter modified luciferases that are about 5 to about 35 fold brighter than wildtype luciferases. Such luciferases are used in accordance with the present invention as stand alone reporters or in multiplexed luciferase assays in combination with one or more other luciferases. As will be appreciated, combinations of luciferases for multiplexed assays of the invention can include both wildtype and modified luciferases.

In further aspects, the present invention provides assay compositions for measurement of modified luciferases of the invention as single luciferase assay formats. In further aspects of the invention, assay compositions are provided that enable simultaneous measurement of at least two different reporters in cell lysates or supernatants using a single assay solution. The luciferase activities of multiple reporters are analyzed by exploiting spectral differences in the emission maxima of the different luciferases.

Improved luciferases used in the present invention include without limitation: (i) a red-emitting firefly luciferase (Red-Fluc) from the Italian firefly Luciola Italica (emission max 609 nm), including intracellular (non-secreted) variants and secreted variants generated by fusing a chymotrypsinogen secretory signal sequence to the amino terminal end of the luciferase; (ii) a green-emitting firefly luciferase (Green-Fluc) from the Italian firefly Luciola Italica (emission max 550 nm), including intracellular (non-secreted) variants and secreted variants generated by fusing a chymotrypsinogen secretory signal sequence to the amino terminal end of the luciferase; (iii) a Cypridina Luciferase or Vargula luciferase (VLuc) from the marine ostracod Vargula Hilgendorfi, a secreted luciferase (emission max 395 nm or 462 nm depending on the substrate used); (iv) Vargula luciferase that has been modified at the C-terminal end with a KDEL sequence (endoplasmic reticulum retention signal) so that it is expressed intracellularly-VLuc-KDEL; (v) a modified secreted blue-emitting (emission max 480 nm) Renilla luciferase (B-Rluc) which is brighter and more stable than native renilla reniformis luciferase; (vi) a green emitting secreted Renilla luciferase (emission max 535 nm) modified to be secreted by fusing a synthetic secretory signal encoding gene sequence in frame with the gene encoding the green emitting modified of renilla luciferase; (vii) a Gaussia luciferase (emission max 482 nm) either native secreted (Gluc) or modified to be expressed intracellularly (Gluc-KDEL).

Luciferases of the Invention

Modified luciferases of the present invention show increased signal magnitude and stability. In certain embodiments, modified luciferases of the invention show at least a 1, 2, 3, 4, 5, 10, 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000-fold increase in the magnitude of the signal over signals seen with wildtype luciferases.

Modified luciferases of the invention may be intracellular (i.e., not secreted), or they may be modified to be secreted. In further embodiments, modified luciferases of the invention are engineered to further express a secretory signal, general at the amino terminal end. In some embodiments, the secretory signal is a synthetic sequence. In specific embodiments, the synthetic sequence is MLLK VVFA IGCI WQA (SEQ ID NO: 7). In yet further embodiments, the secretory signal is any signal that can induce secretion of the encoded protein, including without limitation an interleukin-2 secretory signal and a chymotrypsinogen secretory signal.

Vargula Luciferases of the Invention

In some aspects, the present invention provides a Cypridina Luciferase or Vargula luciferase (VLuc) from the marine ostracod Vargula Hilgendorfi, which is a secreted luciferase (emission max 395 nm or 462 nm depending on the substrate used).

In further aspects, the present invention provides a modified Vargula luciferase that shows increased signal and stability. In certain embodiments, the modified Vargula luciferase of the invention is human codon optimized to increase expression in mammalian systems. In further embodiments, a modified Vargula luciferase of the invention includes a wildtype or a native human codon optimized luciferase with the last two amino acids have been mutated CQ to SN (S=serine, N=asparagine). In still further embodiments, the present invention provides a mammalian vector expressing modified human codon optimized Vargula luciferase expressing intracellular Vargula luciferase. This sequence is the same as the wildtype or native human codon Vargula luciferase with the last two amino acids mutated CQ to SN (S=serine, N=asparagine) and with a KDEL (endoplasmic reticulum retention) sequence added after the C-terminal asparagine residue.

Firefly Luciferases of the Invention

In some aspects, the present invention provides a red-emitting firefly luciferase (Red-Fluc) from the Italian firefly Luciola Italica (emission max 609 nm) and a green-emitting firefly luciferase (Green-Fluc) from the Italian firefly Luciola Italica (emission max 550 nm

In further embodiments, the present invention provides human codon optimized sequences of red-emitting L. Italica luciferases. Such human codon optimized red-emitting L. Italica luciferases show significantly increased activity over wildtype red-emitting L. Italica luciferases (see FIG. 8). In still further embodiments, the present invention provides human codon optimized sequences of red-emitting L. Italica luciferases according to the sequence provided in FIG. 30 (SEQ ID NO: 3). In still further embodiments, the present invention provides human codon optimized sequences of red-emitting L. Italica luciferases encoded by polynucleotides with about 80%-99% sequence identity to SEQ ID NO: 3. In still further embodiments, the present invention provides luciferases that are encoded by polynucleotides with about 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 3.

In still further embodiments, the present invention provides secreted red-Italica luciferases. FIG. 10 shows a comparison of luciferase activity of a human codon optimized red-emitting L. Italica luciferases fused to a chymotrypsinogen secretory signal to a non-secreted form of the human codon optimized red-emitting L. Italica luciferase. As discussed above, a number of different secretory signals can be used to produce secreted forms of modified luciferases of the invention. However, for red firefly luciferase, not all secretory signals produce a secreted luciferase. For example, popular signal sequences such as the N terminal 16 amino acid sequence of Gaussia luciferase and the Interleukin 2 secretory sequence do not successfully produce a secreted form of red emitting firefly luciferase.

Fusing a chymotrypsinogen secretory signal to a human codon optimized red-emitting L. Italica luciferases did successfully produce a secreted form of this luciferase. In some embodiments, the present invention provides a red firefly luciferase (also referred to herein as “red-emitting luciferase” and “red-emitting L. Italica luciferase”) that is modified to include a synthetic secretory signal. In certain embodiments, the modified red firefly luciferase is encoded by the polynucleotide has the sequence provided in FIG. 4 (SEQ ID NO: 2). In still further embodiments, the present invention provides a luciferases encoded by polynucleotides with about 80%-99% sequence identity to SEQ ID NO: 2. In still further embodiments, the present invention provides luciferases that are encoded by polynucleotides with about 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 2. The underlined portion of FIG. 4 is the secretory signal. FIG. 11 shows a comparison of luciferase activities in supernatants and lysates of HEK293 cells transfected with a secreted red Italica Luciferase of the invention.

In further embodiments, the present invention provides human codon optimized sequences of green-emitting L. Italica luciferases. Such human codon optimized green-emitting L. Italica luciferases show significantly increased activity over a previously described thermostable mutant of green-emitting L. Italica luciferase (B. R. Branchini et al., Analytical Biochemistry, 361 (2): 253-262 (2007)—see FIG. 9). In still further embodiments, the present invention provides human codon optimized sequences of green-emitting L. Italica luciferases according to the sequence provided in FIG. 31 (SEQ ID NO: 4). In still further embodiments, the present invention provides human codon optimized sequences of luciferases encoded by polynucleotides with about 80%-99% sequence identity to SEQ ID NO: 4. In still further embodiments, the present invention provides luciferases that are encoded by polynucleotides with about 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 4.

Renilla Luciferases of the Invention

In some aspects, the present invention provides a modified efficiently secreted blue-emitting (emission max 480 nm) Renilla luciferase (B-Rluc), which more stable than the wildtype renilla reniformis luciferase, and a green emitting secreted Renilla luciferase (emission max 535 nm) modified to be secreted by fusing a synthetic secretory signal encoding gene sequence in frame with the gene encoding the green emitting modified of Renilla luciferase. Mammalian cells transfected with the secreted green Renilla luciferase mutant described here show approximately 35-fold higher luciferase activity compared to mammalian cells transfected with the native (human codon optimized) Renilla luciferase (see FIG. 7). Further the secreted green Renilla luciferase shows excellent stability of the bioluminescent signal (without compromising signal intensity) when assayed using the Renilla luciferase assay reagent described in this application (with the stabilizer included, see FIG. 7), thus making it an ideal reporter for High throughput screening applications.

In certain embodiments, the present invention provides a green-emitting Renilla luciferase plasmid sequence with the sequence pictured in FIG. 3 (SEQ ID NO: 1).

Gaussia Luciferases of the Invention

In some aspects, the present invention provides a Gaussia luciferase (emission max 482 nm) that is either native secreted (Gluc) or modified to be expressed intracellularly (Gluc-KDEL). Such Gaussia luciferases can be used in single, double and triple reporter assays as discussed in further detail herein in combination with any of the other luciferases discussed herein or known in the art.

Luciferase Assays of the Invention

In certain aspects, the present invention provides compositions that improve stability and signal for assays utilizing wildtype and/or modified luciferases of the present invention.

In some embodiments, sodium chloride is added to improve the stability of luciferase assays of the invention. In such embodiments, a concentration of sodium chloride is utilized that improves the stability of the bioluminescent signal without affecting intensity. In further embodiments, sodium chloride concentrations in the range of about 0.05 M to about 1 M are used to improve stability of luciferase assays of the invention. In still further embodiments, sodium chloride concentrations of about 0.05 to about 0.5, 0.1 to about 0.4, about 0.2 to about 0.3, and about 0.05 to about 0.2M are used in luciferase assays of the invention. In specific embodiments, sodium chloride is added to improve the stability of assays utilizing wildtype and/or modified Vargula luciferases.

In further embodiments, certain luciferase substrates are added to luciferase assays to improve the stability of the bioluminescent signal. In such embodiments, the substrate added as a stabilizer may be an additional substrate that is not the substrate upon which the luciferase itself acts. For example, in assays utilizing Cypridina luciferase, coelenterazine is added to the assay to stabilize the assay stability. Coelenterazine is an oxidizable luciferin that is easily prone to oxidation but is not a substrate for the Cypridina luciferase. As will be appreciated, any luciferase assay described herein can be further modified by adding substrates for other luciferases as a stabilizer.

In some embodiments, the concentration of luciferase substrate is adjusted to improve the magnitude and/or stability of the signal. In further embodiments, low (under 1 μM) concentrations of substrate is used to improve luciferase signals. For example, for Cypridina luciferase assays, about 1 to about 25 nM Vargulin are used in assays of the invention. In further embodiments, about 1-100, 5-90, 10-80, 15-70, 20-60, 25-50, and 30-40 nM Vargulin are used in assays of the invention. In further exemplary embodiments, substrates for the luciferase assays described herein (including Cypridina, Gaussia and L. Italica luciferases) are added in concentrations of from about 1 nM to about 250 μM. In still further embodiments, substrates are added in concentration of about 10 nM-200 μM, 50 nM-150 μM, 100 nm-100 μM, 150 nm-50 μM, 200 nM-25 μM, 300 nM-10 μM, 500 nM-1 μM.

In some embodiments, Gaussia luciferases of the invention are used with optimized reagents to produce increased activity. Kinetics of the Gaussia luciferase assay using the GAR-1 reagent is shown in FIG. 14. Measurement of the luciferase activity in supernatants of cells (transfected with Gaussia luciferase) using GAR-1 reagent from Targeting systems showed increased activity from Renilla luciferase assays from another vendor. The data in FIG. 14 is presented as an average of triplicate determinations measured on a Turner TD2020 luminometer. GAR-1 reagent has been described in detail in US Pat Appl Publ 2008074485, which is hereby incorporated by reference in its entirety and in particular for all teachings related to assay reagents for the Gaussia luciferase assay.

In certain embodiments, Gaussia luciferase assays of the invention utilize reagents stabilized with stabilizing agents. In one non-limiting example, the stabilizing agents include NP40 (Sigma) and/or coelenterazine. In certain embodiments, about 5 to about 200 μM coelenterazine is used. In still further embodiments, about 10-150, 20-125, 30-100, 40-75, 50-60 μM coelenterazine is used. In yet further embodiments, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μM coelenterazine is used. Stability of Gaussia luciferase assays using the GAR-2 reagent are shown in FIG. 15. Using the GAR-2B version of the Gaussia luciferase assay reagent, the bioluminescent signal remains very stable (FIG. 15A) In the absence of the stabilizer, the signal intensity is a little higher initially but decays faster than in the presence of the stabilizer (FIG. 15B). Note that the data presented in FIG. 15A and B is an average of triplicate determinations measured on a Turner TD2020 luminometer. The GAR-2 and GAR-2B reagents are stabilized versions of the GAR-1 reagent discussed in US Pat Appl Publ 2008074485, which is hereby incorporated by reference in its entirety and in particular for all teachings related to reagents for Gaussia luciferase assays. The GAR-2 reagent includes the composition GAR-1 with an additional 30 μM coelenterazine. GAR-2B reagent includes the composition GAR-1 with and additional 75 μM coelenterazine. Without being limited by theory, it is possible that the higher (approximately 3-fold) signal intensity seen with the GAR-2B reagent is due to the higher concentration of coelenterazine. FIG. 12B shows the stability of the Gaussia luciferase with the GAR-2 reagent including a stabilizer.

In certain embodiments, stability of firefly luciferase assays is improved using FLAR-1 reagents (Targeting Systems). FIG. 16 shows the results from experiments using the FLAR-1 reagent from Targeting Systems. In the experiments shown in FIG. 16, the FLAR-1 reagent was added to the supernatant cell culture media.

Dual and Triple Luciferase Assays

In some aspects, the present invention provides dual luciferase assays based on spectral resolution of two or more different luciferases. As will be appreciated, these assays can include different wildtype luciferases, different modified luciferases, or a mixture of a wildtype and a modified luciferase. Such assays rely on differences in the emission spectra of the reporters used. In further embodiments, reagents are modified to allow for more efficient multiplexing. For example, when Gaussia luciferases are multiplexed with firefly luciferases, EDTA is omitted from the reaction mixture to allow efficient reporter activity.

FIG. 13A shows the emission spectra of a dual reporter assay utilizing a Vargula and Red Italica luciferase of the invention. The luciferases were expressed in samples of transfected cells. The luciferases used in the experiments pictured in FIG. 13A represent a modified red emitting firefly luciferase of the invention that is human codon optimized and intracellular (non-secreted) and a Cypridina luciferase of the invention that is from Cypridina hilgendorfi modified to be human codon optimized and secreted.

FIG. 13B shows the emission spectra of a triple reporter assay utilizing Vargula, Green Renilla and Red Italica luciferases. These emission spectra were in samples of transfected cell lysates. The Vargula and red-emitting firefly luciferases are those as described above for FIG. 13A and the Green Renilla luciferase is an improved secreted Green luciferase mutant as described in further detail herein.

All patents and other references cited in the specification are indicative of the level of skill of those skilled in the art to which the invention pertains, and are incorporated by reference in their entireties, including any tables and figures, to the same extent as if each reference had been incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the present invention is well adapted to obtain the ends and advantages mentioned, as well as those inherent therein. The methods, variances, and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

Also, unless indicated to the contrary, where various numerical values or value range endpoints are provided for embodiments, additional embodiments are described by taking any 2 different values as the endpoints of a range or by taking two different range endpoints from specified ranges as the endpoints of an additional range. Such ranges are also within the scope of the described invention. Further, specification of a numerical range including values greater than one includes specific description of each integer value within that range.

Thus, additional embodiments are within the scope of the invention and within the following claims.

EXAMPLES Example 1 Transfection of Mammalian Cells with Modified Luciferases

HEK-293 cells were grown in DMEM/10% FBS (fetal bovine serum) and transfected with plasmids expressing either the human codon-optimized or non-human codon optimized forms of the red emitting and green emitting firefly luciferases (from Luciola Italica) under control of the CMV promoter. Transfections were performed using the Targefect F-2 reagent (Targeting Systems) using the manufacturers protocols. Forty eight hours post transfection, the cells were lysed using the cell lysis reagent (CLR-1) from Targeting Systems, Santee. 20 μl aliquots of the cell lysate were mixed with 100 μl of the FLAR-1 (firefly luciferase assay reagent from Targeting Systems).

Example 2 Cypridina Luciferase Assays with Increased Stability

Compositions were developed for achieving optimal performance of Cypridina luciferase assay reagents. These assays had improved stability of the bioluminescent signal without affecting the overall activity of the enzyme.

Vargulin is generally unstable and easily oxidized, making long term storage of this substrate difficult. However, Vargulin stored in an acidic buffer (66 mM monobasic potassium phosphate, pH 6-6.5) and stored at −80° C. was very stable and did not lose activity even when stored for several months. In contrast, Vargulin dissolved in a neutral to basic phosphate buffer (e.g. 200 mM dibasic potassium phosphate (ph 8)) is very unstable and begins to lose activity rapidly within a few hours at room temperature. Cypridina luciferase activity was optimal when 200 mM dibasic potassium phosphate was used as the reaction buffer instead of 66 mM monobasic sodium phosphate. Hence 200 mM dibasic potassium phosphate was used as the reaction buffer. Concentrations of be 3-6 nM Vargulin were found to be effective, and these concentrations are much lower than what is generally used in such assays (see for example Wu et al (2007) Biotechniques, 42(3):290-292).

The Cypridina luciferase assay showed increased stability when sodium chloride was included in the reaction. For example, FIG. 1 shows the relative luciferase stability (RLS) between VLAR-1 (no sodium chloride) and VLAR-2 (VLAR-1+sodium chloride). Sodium chloride clearly stabilized the RLS. For the experiments in FIG. 1, 20 μl of sample was added with 40 μl of VLAR solution for the assay followed by 20 μl of Vargulin substrate.

FIG. 5 shows a further titration experiment indicating that sodium chloride concentrations of around 0.5M provide increased stability over control reagents with no sodium. Further concentrations that are of use in stabilizing such assays include from about 25 mM to about 750 mM sodium chloride. For experiments in FIG. 5, 5 μl of the indicated concentrations of sodium chloride solutions were added to 35 μl of VLAR buffer (20 mM dibasic potassium phosphate, pH=8.0). The assay was carried out by mixing 20 μl of sample with 40 μl of VLAR buffer (with sodium chloride) and then adding 20 μl of Cypridina luciferin.

Further stability of the bioluminescent signal as well as improvement in overall luciferase activity was observed when coelenterazine, another oxidizable luciferin easily prone to oxidation (but not a substrate for Cypridina luciferase) m was included in the assay composition. A 15 minute pre-incubtion was found to result in increased stability of the bioluminescent signal using sample volumes between 5 and 20 μl (roughly 40% drop in 26 minutes using an assay volume of 20 μl and 15% drop in 26 minutes using an assay volume of 5 μl—see FIGS. 2A and 2B. A concentration of coelenterazine that worked well to stabilize the reagent was 15 μM. Concentrations in the range of about 10 μM to about 50 μM can also be used. The inclusion of coelenterazine in the composition decreased the background of the assay by more than 10-fold (background reading dropped form 153.6 to 12.4) and also resulted in a 15% increase in the intensity of the bioluminescent signal. Controls in which buffers with identical composition (i.e., inclusion of coelenterazine but omission of Cypridina luciferin) showed no activity. Coelenterazine is not a substrate for Cypridina luciferase and can be used to safely reduce the background and increase stability when Cypridina luciferase is assayed alone or in combination with other luciferases (such as firefly luciferase) which do not use coelenterazine as a substrate. For the experiments shown in FIG. 2, 5 or 20 μl of the sample (media supernatant) was mixed with 40 μl of the VLAR buffer (200 mM dibasic potassium phosphate, 50 mM NaCl). The firefly and Cypridina luciferase assay reagents can be mixed into a single solution which can be used to efficiently measure both Cypridina luciferase and firefly luciferase activity by spectrally resolving the luciferases using appropriate filers. However, the DTT concentration in the firefly luciferase assay reagent can affect activity in such situations, because the activity of both luciferases is decreased due to interference of DTT (present in low concentration in the firefly assay reagent with the Cypridina luciferase assay (there is almost a 10-fold drop in Cypridina luciferase activity). However, since the signal intensity of the Cypridina luciferase assay is very robust, the signal is still acceptable and improvement in Cypridina luciferase activity is observed if the DTT concentration in the firefly luciferase assay reagent is dropped to 2.5 mM (a 3 fold drop in activity of Cypridina luciferase is still observed). Single solution based dual assays in which Cypridina luciferase is multiplexed with Green emitting Renilla luciferase work very well without loss of activity of either Cypridina or renilla luciferase when the two solutions are mixed.

Example 3 Renilla Luciferase Assays Utilizing Modified Renilla Luciferases and Stabilizing Reagents

The secreted modified green Renilla luciferase of the present invention showed significantly greater activity over wildtype Renilla luciferase—see FIG. 7. For the experiments pictured in FIG. 7, HEK 293 cells were transfected with expression vectors expressing either native Renilla luciferase or the secreted Green Renilla luciferase mutant. Cells were lysed 48 hrs post transfection and assayed for luciferase activity.

Assays with and without stability assay reagents for green Renilla luciferase were investigated. FIG. 6 shows that assays conducted with stabilizer showed greater stability than those without. The composition of the Renilla luciferase assay reagent (no stabilizer) was: 30 μM coelenterazine, 0.4×PBS (Ca, Mg free), 0.027% NP40. The composition of the Renilla luciferase assay reagent (with stabilizer) was: 30 μM coelenterazine, 0.4×PBS (Ca, Mg free), 0.227% NP40. Stabilizer is 2% NP40 (a non-ionic detergent).

Example 4 Kinetics of Different Luciferases

Reactions were set up to measure the kinetics of the luciferase activities of different luciferases in samples of transfected cells. Luciferase activities were assayed using the luciferase assay reagents supplied with the LiveResponse assay kit. These data are shown in FIG. 12A: Red Luciola (firefly), luciferase, FIG. 12B Gaussia Princeps luciferase (this is FIG. 15C), FIG. 12C: Cypridina luciferase, and FIG. 12D: Green Renilla luciferase. Data represents mean of triplicate determinations.

Example 5 Comparison of Expression Vectors Expressing Modified Vargula Luciferases

Transfection protocols were as follows: HEK-293 cells were grown in DMEM/10% FBS (fetal bovine serum) and transfected with plasmids expressing wither the human codon-optimized to non-human codon optimized forms of native VLuc, HC-VLuc, sequence 1) or modified HC-VLucs under control of the CMV promoter. Transfections were performed using the Targefect F-2 regent (Targeting Systems) using the manufacturers protocols.

The stability of the bioluminescent signal of Cypridina Luciferase assessed using supernatants of HEK293 cells transiently transfected with the pCMV VLuc expression vector is shown in FIG. 17.

In FIG. 19, the stability of the bioluminescent signal of Cypridina Luciferase was assessed using supernatants from HEK 293 cells transiently transfected with the pCMV-VLuc expression vector. Samples were assayed using the VLAR-2 (VLAR-1 reagent from Targeting Systems with sodium chloride) of the Cypridina luciferase assay reagent.

Human codon optimization of the gene sequence encoding the VLuc led to a 5-fold improvement of luciferase expression in HEK-293 transfected with expression vectors containing the human codon optimized versions of the vargula luciferase genes compared to the native sequences (i.e. Non-human codon optimized sequences). Addition of the KDEL sequence at the C-terminal end results in intracellular expression of VLuc.

Example 6 Construction of Blue-Emitting (Blue Shifted) and Green Emitting Mutants of Secreted Renilla Luciferase for Use as Secreted Reporters in Single or Multiplexed Luciferase Assays

A synthetic signal peptide was deduced by rational design: MLLK VVFA IGCI VVQA (SEQ ID NO: 7). The sequence of this signal peptide was based on rational design using signal sequences from the secretory signals known in the art, including those available at: http://www.unitargeting.com/Resources/Trends07.pdf

Blue-Sifted Secreted Renilla Luciferase Mutants

Secreted mutants were constructed containing signal peptide fused to amino terminal region of the human codon optimized renilla reniformis luciferase with the following additional mutations which enable i) efficient refolding after secretion to obtain an active form of the enzyme (Loma Linda paper, cysteine 124 was mutated to alanine) and additional mutations to cause a shift in the emission max of Renilla luciferase. MLLK VVFA IGCI VVQA-HCRLuc with following mutations C124A; N53Q; V146M. Emission maxima=475 nm

Secreted BLuc Sequence 2: MLLK VVFA IGCI VVQA-HCRLuc with following mutations C124A; N53Q; V146M and the following eight additional mutations A55T, S130A, K136R, A143M, M185V, M253L, S287L. The 8 additional mutations increase intensity of the bioluminescent signal (Emission Maxima 475 nm)

Red Shifted Renilla Luciferase Mutants:

Secreted RLuc Sequence 1: MLLK VVFA IGCI VVQA-HCRLuc with following mutations C124A, D162E

Secreted RLuc Sequence 2: MLLK VVFA IGCI VVQA-HCRLuc with following mutations C124A; and the following eight additional mutations AI23S/D154M/E155G/D162E/I163L/V185L F262W. Emission Maxima 535 nm

Secreted RLuc Sequence 3: MLLK VVFA IGCI VVQA-HCRLuc with following mutations C124A; and the following eight additional mutations AI23S/D154M/E155G/D162E/I163L/V185L. Emission Maxima 535 nm

Example 7 Tests for Developing Assays for Vargula Luciferase

In some embodiments, different buffer solutions are used to improve assays utilizing wildtype and/or modified luciferases of the invention. In certain embodiments, a 1:1 mixture of 0.1 M Tris HCl and 75 mM sodium phosphate is used as the assay buffer.

Several different parameters were tested to develop an assay for vargula luciferase:

Effects of using either an acidic buffer (e.g., potassium phosphate pH 5-6.8), Tris HCl pH 7.4, Tris phosphate buffer pH (8-8.5) as well as varying assay volumes were tested. In general the use of acidic conditions significantly reduced the intensity of the bioluminescent signal (typically 5-10 fold) while increasing the stability somewhat. Using Tris HCL ph 7.4, the activity as the assay buffer resulted in 5-10 fold brighter bioluminescence but the luminescent signal was highly unstable.

Use of a buffer mixture (1:1) of 50 mM Tris HCl, pH 7.4 and 100 mM dibasic sodium phosphate resulted in improved stability of the bioluminescent signal without compromising the intensity of the bioluminescent signal. An interesting finding was that inclusion of 0.2 M NaCl further increased stability of the bioluminescent signal. Lastly the amounts of Vargulin needed for optimal activity using this buffered condition are very low (1-10 nM range) making the assay extremely useful and economical.

Increasing the concentration of Vargulin further did not increase stability of the assay further.

Stock Vargulin substrate solutions stored in an acidic condition pH (5.5-6) were relatively stable over several months when stored at −80° C.

Other parameters tested: Other stabilizers such as DTT (dithiothreitol), detergents like NP-40 or EDTA were unable to increase the intensity of the luminescent signal or improve stability of the assay. EDTA decreased the VLuc activity by at least 5-fold.

Thus one aspect of the invention concerns the following composition and variations thereof: 20 μl of cell supernatant assays with 50 μl of Tris/phosphate buffer, pH 8, 0.2 M NaCl, 10 μl of 5-100 nM vargulin in 66 mM potassium phosphate (monobasic). In certain assays, the effective concentration of vargulin in the assay mix is as low as 20 nM which is approximately 50-fold lower than that reported in the literature (see for example Wu et al (2007) Biotechniques, 42(3):290-292)

Comparison of luciferase activity in cells transfected with vargula luciferase with luciferase activity in cells transfected with firefly luciferases from Photinus pyralis or Luciola Italic showed that vargula luciferase was a much more sensitive reporter (10-20 fold improvement in bioluminescent signal compared to firefly luciferase, assay done in HEK-293 cells, all expression vectors were expressed luciferase under control of the CMV promoter). An exemplary assay protocol included: 20 μl aliquots of Cell supernatants (media with 5% serum) were mixed with 100 μl of assay dilution buffer (50 μl of 50 mM TrisHCl, 100 mM dibasic sodium phosphate, pH 8) and 10 μl of vargulin in sodium phosphate buffer pH 6 (final concentration of vargulin in reaction mix 10-25 nM). The sample was mixed well and bioluminescent activity was recorded in a Turner TD2020 luminometer integrated over a 20 sec time interval.

Example 8 Activity in Cell Supernatant and Cell Lysates of Cell Transfected with Either a Plasmid Vector Expressing Secreted Vargula Luciferase or an Intracellular Form of Vargula Luciferase

In cells transfected with the secreted form of modified vargula luciferase, 80% of the activity was secreted into the cell supernatant and only 20% is cell-associated.

FIG. 18 shows intracellular and secreted Cypridina luciferase activity. Luciferase activity in cell supernatants and cell lysates of cells transfected with a plasmid vector expressing secreted vargula luciferase. As shown in FIG. 18 cells transfected with the secreted form of modified vargula luciferase, 80% of the activity is secreted into the cell supernatant and only 20% is cell-associated.

In cells transfected with vargula luciferase modified at the C-terminal end with a KDEL sequence, approximately 95% of the activity was intracellular and 5% is secreted.

Example 8 Development of a Dual Reporter System Based on Blue and Red Shifted Mutants of Secreted Renilla Luciferase

Secreted mutants: Secreted mutants were constructed containing signal peptide fused to amino terminal region of the human codon optimized renilla reniformis luciferase with the following additional mutations which enable i) efficient refolding after secretion to obtain an active form of the enzyme (Cysteine 124 was mutated to alanine) and additional mutations to cause a shift in the emission max of renilla luciferase: MLLK VVFA IGCI VVQA-HCRLuc with following mutations: C124A; N53Q; V146M. Emission maxima=475 nm

Secreted RLuc Sequence 2: MLLK VVFA IGCI VVQA-HCRLuc with following mutations. C124A; N53Q; V146M and the following eight additional mutations A55T, S130A, K136R, A143M, M185V, M253L, S287L. The 8 additional mutations increase intensity of the bioluminescent signal. Emission Maxima 475 nm

RED SHIFTED RENILLA LUCIFERASE MUTANTS: Secreted RLuc Sequence 1: MLLK VVFA IGCI VVQA-HCRLuc with following mutations: C124A, D162E

Secreted RLuc Sequence 3: MLLK VVFA IGCI VVQA-HCRLuc with following mutations: C124A; and the following eight additional mutations AI23S/D154M/E155G/D162E/I163L/V185L F262W. Emission Maxima 535 nm

Secreted RLuc Sequence 4: MLLK VVFA IGCI VVQA-HCRLuc with following mutations: C124A; and the following eight additional mutations. A123S/D154M/E155G/D162E/I163L/V185L. Emission Maxima 535 nm

A single solution dual luciferase assay based on secreted renilla luciferase blue emitting (emission max at 475 nm) and green emitting mutants (emission max at 535 nm).

The mutations in the above sequences lead to the efficient expression of secreted renilla luciferase in the transfected cells. The two luciferases can therefore be used in combination as a dual reporter system and the luciferase activity of each luciferase in the transfected cells can be resolved by using appropriate filters. The reagent compositions for renilla luciferase assay reagents are described Walia, US Pat Appl Publ 2008074485, entitled Enhancing a Luminescent Signal, which is incorporated herein by reference in its entirety and in particular for all teachings related to Renilla luciferase assay reagents.

Example 9 Development of a Triple Reporter System Based on Red and Green Emitting Firefly Luciferases and Gaussia Luciferase/Renilla Luciferase

Composition of the Gaussia luciferase assay reagent (GAR-1) has been described in detail in a US Pat Appl Publ 2008074485, which is hereby incorporated by reference in its entirety and in particular for all teachings related to assay reagents for the Gaussia luciferase assay. An assay reagent useful for simultaneous measurement of all there reporters in a single solution was designed by omitting EDTA from the composition of the Gaussia luciferase assay reagent and then including all the ingredients necessary for assay of firefly luciferase in a single composition. The rationale behind this is that the EDTA interferes with the firefly luciferase assay (magnesium is an important co-factor for firefly luciferase and EDTA chelates magnesium). The ingredients required for Firefly luciferase assay included in the assay composition were as follows—ATP, DTT. Firefly luciferin, magnesium sulfate, magnesium bromide (helps increase brightness of luminescent signal) and phosphate buffer.

The composition of the single solution for a triple reporter assay for measuring Gaussia luciferase or Renilla luciferase in combination with red and green emitting firefly luciferase is as follows:

-   -   0.1×PBS. 5.4 ml of 5% NP40 diluted to 1000 ml and add the         following:     -   To 800 ml of the above solution add the following:     -   Tricine 3.227 g (20 mM)     -   1M Magnesium sulfate 0.7H2O 2.51 ml (2.67 mM     -   Magnesium bromide 0.6 H2) (1.07 mM)—add 2.14 ml of 500 mM stock         solution     -   25 mM OTT (3.86 g)     -   530 μM ATP (2.72 g)     -   CoA (0.18 g)—optional     -   Adjust with sodium phosphate to pH 7.8     -   Add 940 μM D-Luciferin (fee acid)—253.81 mg     -   CDTA-0.8289 g     -   940 μM D-luciferin (free acid)—253.81 mg     -   CDTA-0.8289 g     -   0.8M Tris (0.02 M EDTA)—43.53 ml     -   Add GAR reagent without EDTA to a total volume of 1 liter     -   Dilute 100× coelenterazine substrate with the above solution to         1× just before use. Use normal 3 mg/5 ml absolute alcohol         acidified with 30 μl of 2N HCl)         NOTE: This assay reagent does not contain enough cell lysis         reagents. Hence cells have to be first lysed using 1× Cell Lysis         Buffer (compatible with use of all luciferases (prepared from 5×         stock solution described below:         Dilute the 5× Cell lysis buffer described below with water to 1×         concentration and add to washed cells and shake at 400 rpm for         20 mins to lyse cells.

Composition of 5× Cell Lysis Buffer:

For 1 liter of Buffer

-   -   5 ml NP 40 (undiluted)     -   25 ml Tris HCl ph 8     -   1.45 g NaCl     -   50 ml glycerol

Example 10 Development of a Single Solution Triple Luciferase Reporter Assay Based on Red and Green Emitting Firefly Luciferases and Vargula Luciferase

A vargula luciferase-based triple reporter system was prepared by first preparing the vargula luciferase assay reagent (VLAR-1) and mixing it in a 1:1 ratio with the firely luciferase assay reagent (FLAR-T) to give the triple assay reagent TVLAR-1.

Assay protocol: To 20 μl of cell lysate add 100 μl of the TVLAR-1 reagent and read in the Victor luminometer (Perkin Elmer) or Varian (Promega) using appropriate filters.

Preparation of VLAR-1 Reagent:

Composition of the Vargula Luciferase Assay reagent is described below

-   -   500 ML OF 0.1 M TRIS HCL PH 8     -   500 ML of dibasic sodium phosphate 200 mM     -   200 ml of 5 nM Vargulin in 66 mM potassium phosphate pH 5.5     -   pH of final solution is 8-8.5

Composition of the FLAR-T Reagent

SOLUTION A: 0.1×PBS. 5.4 ml of 5% NP40 diluted to 1000 ml and add the following:

-   -   To 800 ml of the above solution add the following:     -   Tricine 3.227 g (20 mM)     -   1M Magnesium sulfate. 7H2O 2.51 ml (2.67 mM)     -   Magnesium bromide (0.6 H2) (1.07 mM)—add 2.14 ml of 500 mM stock         solution     -   5 mM DTT (in some embodiments, any range between 5 mM and 30 mM         can be used, including 5, 10, 15, 20, 25, 26, 27, 28, 29, and 30         mM)     -   530 μM ATP (2.72 g)     -   CoA (0.18 g)—optionally omitted     -   Adjust with sodium phosphate to pH 7.8     -   Add 940 μM D-Luciferin (free acid)—253.81 mg     -   CDTA-0.8289 g     -   940 μM D-luciferin (free acid)—253.81 mg     -   CDTA-0.8289 g     -   941 μM D-luciferin (free acid)−253.81 mg     -   CDTA-0.8289 g     -   0.8M Tris (0.02 M EDTA)—43.53 ml         ADD SOLUTION A to a total volume of 1 liter

NOTE: This assay reagent does not contain enough cell lysis reagents for effective lysis. Hence cells should first be lysed, e.g., using 1× Cell Lys is Buffer (compatible with use of all luciferases (prepared from 1× stock solution described below: Dilute the 5× Cell lysis buffer described below with water to 1× concentration and add to washed cells and shake at 400 rpm for 20 mins to lyse cells.

Composition of 5× cell lysis buffer:

-   -   For 1 liter of Buffer     -   5 ml NP 40 (undiluted)     -   25 ml Tris HCl ph 8     -   1.45 o NaCl     -   50 ml glycerol

Composition of Firefly luciferase assay reagent (for use of firefly luciferase as a single reporter gene).

-   -   20 mM tricine (179.2 3.55 g)     -   MgCo3 1.07 mM 0.55 g     -   Magnesium sulfate 2.7 mM (277 ml)     -   0.1 mM EDTA     -   20 mM DTT (4.25 g)     -   530 μM ATP (3 g)     -   CoA (0.198 g)     -   Add disodium phosphate 25 g to ph 7.8     -   Add 793 ml water before pH     -   470 μM D Luciferin free acid 279.2 mg     -   5×CCLR 307 ml

Composition of 5×CCLR:

-   -   0.8 M Tris 0.02 M EDTA pH 8-156 ml     -   Glycerol 500 ml     -   Triton X100 50 ml     -   CDTA-7.5 m moles (2.7 g)     -   DTT 10 mM 1.542 g total vol 1 liter.

Example 11 Development of a Single Solution Triple Luciferase Reporter Assay Based on Red and Green Emitting Firefly Luciferases and Vargula Luciferase

Addition of stabilizer does not significantly affect (i.e, there is very little decrease in signal intensity) intensity of bioluminescent signal of Renilla luciferase in supernatants and lysates. FIG. 20 (top panel) shows a Renilla assay performed with 10 μl of Renilla Lysate and 20 μl of Renilla Supernatant. Assay went as follows: 20 or 10 μl of sample (Supernatant or Lysate), 50 μl of RLAR-1 reagent (Targeting Systems). FIG. 20 (bottom panel) shows Renilla Assay was performed using the same volumes of lysate and supernatant as in the experiments in the top panel. Assay protocol was as follows: 10 or 20 μl of lysate or supernatant depending on the assay, 50 μl of the RLAR-1 reagent and an additional 8 μl of RLAR stabilizer for an increased stability profile for a time course reading. The stabilizer lowered the initial RLU reading (decreased from approximately 9000 to approximately 7000 rlu) but showed a much higher level of stability when observed over 30 minutes to 1 hour (FIG. 12C). The RLAR-1 reagent is useful for high throughput screening (HTS) applications in which a large number of samples need to be assayed. In the absence of the stabilizer, the signal intensity decays faster than in the presence of stabilizer (FIG. 21). Note: Data presented is average of triplicate determinations measured on a Turner TD2020 luminometer. In FIG. 21, a time course was taken using the standard protocol of 10 μl lysate, 50 μl of RLAR reagent without stabilizer indicating drop in Renilla luciferase activity.

FIG. 22 shows the stability of the bioluminescent signal of Cypridina luciferase and firefly luciferase using the DLAR-3 reagent. This reagent is useful for HTS applications involving both Cypridina luciferase and the red-emitting Luciola luciferase. Note: Data presented is average of triplicate determinations measured on a Turner TD2020 luminometer. The DLAR-3 reagent (Targeting Systems) is a dual assay reagent based on secreted Cypridina luciferase and a secreted or intracellular red-emitting firefly luciferase.

FIG. 28 shows emission spectra of Cypridina and Firefly luciferases in samples of transfected cells (lysates or supernatants). The emission spectra were recorded on a Fluorolog-3 spectrofluorometer (Horiba Scientific, Japan) using a liquid nitrogen cooled CCD. The luciferases were assayed by mixing 200 ul of the sample with the appropriate luciferase assay reagent to obtain spectral profiles. Emission max of Cypridina Luciferase is 463 nm; Red Italica 617 nm.

Example 12 Double and Triple Luciferase Reporter Assays Based on Renilla Luciferase, Firefly Luciferase and Vargula Luciferase

Kinetics of luciferase activity of different luciferase reporters using luciferase assay reagents in the DLAR-5 system are shown in FIG. 23. Reactions were set up to measure the kinetics of the luciferase activities of different luciferases in samples of transfected cells. Luciferase activities were assayed using the DLAR-5 luciferase assay reagents. The decay of the renilla luciferase signal shown in Panel B above can be greatly minimized (ie the bioluminescent signal can be rendered much more stable by addition of a Renilla luciferase stabilizer to the DLAR-5 buffer.

FIG. 24 shows Emission spectra of different luciferases in samples of transfected cell lysates. Relative luciferase activities of Cypridina, Green Renilla luciferases were assayed with the appropriate luciferase assay reagent to obtain spectral profiles. The emission max of Vargula luciferase is 463 nm; Green Renilla luciferase is 527 nm. Note that the data presented in this application is performed with the green-emitting mutant that emits at 527 to 530 nm (this is the variation in emission maxima seen and the luciferase is different in sequence, properties and emission maximum from the 535 nm emitting intracellular green emitting Renilla luciferase mutant described in US Patent Publication No. 20090136998, which is hereby incorporated by reference in its entirety and in particular for all teachings related to Green Renilla luciferase.

FIG. 25 shows kinetics of luciferase activity of different luciferase reporters using luciferase assay reagents in the triple reporter system. Reactions were set up to measure the kinetics of the luciferase activities of different luciferases in samples of transfected cells. Luciferase activities were measured using the TLAR luciferase assay reagents (Targeting Systems).

FIG. 26 shows emission spectra of different luciferases in samples of transfected cell lysates. Relative luciferase activities of Cypridina, Renilla and Red Luciola Italia luciferases were assayed with the appropriate luciferase assay reagent to obtain spectral profiles. The emission max of Vargula luciferase is 463 nm; Green Renilla luciferase is 527 nm and Red Luciola Italia luciferase is 617 nm.

The present invention also provides a single solution-based triple luciferase reporter assay involving Cypridina luciferase multiplexed with Green-emitting Renilla luciferase and Red-emitting Firefly luciferase. This assay is compatible with high throughput applications. This assay is also optionally in a format where the three luciferases can be assayed separately using three different assay reagents 

What is claimed is:
 1. A multiplexed luciferase assay composition comprising: multiple luciferase reporters, wherein at least two of the luciferase reporters are selected from the group consisting of a firefly luciferase, a Renilla luciferase, a Gaussia luciferase and a Cypridina (Vargula) luciferase, and wherein the different luciferase reporters emit at different wavelengths and/or utilize different substrates, and wherein the firefly luciferase is a red-emitting human codon optimized luciferase encoded by SEQ ID NO:3 with an emission maximum of approximately 617 nm; or wherein the firefly luciferase is a green-emitting human codon optimized luciferase encoded by SEQ ID NO:4 with an emission maximum of approximately 550 nm; or wherein the Renilla luciferase comprises A55T, S130A, K136R, A143M, M185V, M253L, and S287L mutations or AI23S, D154M, E155G, D162E, I163L, and V185L mutations compared to wildtype Renilla luciferase; or wherein the Vargula luciferase is encoded by SEQ ID NO: 6; and wherein at least one of the luciferase reporters comprises a secretory signal, wherein the secretory signal is peptide sequence of SEQ ID NO:
 7. 